Prevention of neutrophil recruitment

ABSTRACT

Aspirin (ASA) triggers a switch in the biosynthesis of lipid mediators, inhibiting prostanoid production and initiating 15-epi-lipoxin generation, through the acetylation of cyclooxygenase II.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This is a continuation application of U.S. Ser. No. 11/742,893, filedMay 1, 2007, which is a Divisional application of U.S. Ser. No.11/106,066, filed Apr. 13, 2005, now U.S. Pat. No. 7,227,031, which is acontinuation application of U.S. Ser. No. 10/366,194, filed Feb. 13,2003, now U.S. Pat. No. 6,960,674, which is a continuation of U.S. Ser.No. 10/176,744, filed Jun. 20, 2002, now U.S. Pat. No. 6,720,354, whichis a Divisional of U.S. Ser. No. 09/525,742, filed Mar. 14, 2000, nowU.S. Pat. No. 6,433,202, which claims benefit of U.S. Ser. No.60/125,209 filed Mar. 18, 1999, the contents of which are incorporatedherein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The work leading to this invention was supported by grants GM-38765 andDK-5 0305 for the National Institute of Health. The U.S. Governmenttherefore may have certain rights in this invention.

BACKGROUND OF THE INVENTION

Aspirin (acetylsalicylic acid, ASA) has been available for use as ananalgesic-antipyretic for almost a century and novel therapeuticapplications for this drug, for example in lowering the risk ofmyocardial infarction or as a prophylaxis against colorectal cancer,continue to be uncovered (Weissmann, G. (1991) Sci. Am. 264, 84-90;Ridker, P. M., Cushman, M., Stampfer, M. J., Tracy, R. P. & Hennekens,C. H. (1997) N. Engl. J. Med. 336, 973-979; Marcus, A. J. (1995) N.Engl. J. Med. 333, 656-658). The acetylation of cyclooxygenases I and II(COX I and II) and the subsequent irreversible inhibition ofprostaglandin (PG) and thromboxane biosyntheses are well understoodmechanisms of some of ASA's pharmacological actions (Marcus, A. J.(1995) N. Engl. J. Med. 333, 656-658; Herschman, H. R. (1998) TrendsCardiovasc. Med. 8, 145-150). More recently, ASA was found to cause aswitch in eicosanoid biosynthesis as the acetylation of COX II changesthe enzyme's activity to produce 15R-hydroxyeicosatetraenoic acidfromagonist-released arachidonic acid Herschman, H. R. (1998) TrendsCardiovasc. Med. 8, 145-150). Human neutrophils, and other cellspossessing 5-lipoxygenase, utilize this substrate via transcellularbiosynthetic routes to produce 15-epi-lipoxin A₄ (15-epi-LXA₄) and15-epi-lipoxin B₄ (15-epi-LXB₄) (Serhan, C. N. (1997) Prostaglandins 53,107-137; Chiang, N., Takano, T., Clish, C. B., Petasis, N. A., Tai,H.-H. & Serhan, C. N. (1998) J. Pharmacol. Exp. Ther. 287, 779-790).These aspirin-triggered lipoxins (ATL) are the endogenous 15Renantiomeric counterparts of lipoxin A₄ (LXA₄) and lipoxin B₄ (LXB₄),respectively, and share their bioactivities (Serhan, C. N. (1997)Prostaglandins 53, 107-137(5)).

Unlike other eicosanoids (e.g., leukotrienes, PGs, etc.), which aregenerally considered local pro-inflammatory mediators, lipoxins (LX)display potent inhibitory actions in several key events in inflammation,such as polymorphonuclear cell (PMN) chemotaxis, transmigration acrossendothelial and epithelial cells, and diapedesis from post-capillaryvenules (Serhan, C. N. (1997) Prostaglandins 53, 107-137(5)). LX aregenerated in several pathogenic scenarios in vivo, for example: in lungtissue of patients with severe pulmonary disease; and by PMN frompatients with asthma or rheumatoid arthritis, where their presence isproposed to be linked to long-term clinical improvement (Lee, T. H.,Crea, A. E., Gant, V., Spur, B. W., Marron, B. E., Nicolaou, K. C.,Reardon, E., Brezinski, M. & Serhan, C. N. (1990) Am. Rev. Respir. Dis.141, 1453-1458; Chavis, C., Chanez, P., Vachier, I., Bousquet, J.,Michel, F. B. & Godard, P. (1995) Biochem. Biophys. Res. Commun. 207,273-279; Chavis, C., Vachier, I., Chanez, P., Bousquet, J. & Godard, P.(1996) J. Exp. Med. 183, 1633-1643; Thomas, E., Leroux, J. L., Blotman,F. & Chavis, C. (1995) Inflamm. Res. 44, 121-124). Interestingly, ATLshow an even greater level of inhibition than native LX in preventingneutrophil adhesion, where they are ˜twice as potent (Serhan, C. N.(1997) Prostaglandins 53, 107-137). ATL are also more potent inhibitorsof microbial induction of cytokine release. Specifically, 15-epi-LXA₄showed greater inhibition than LXA₄ of S. typhimurium-induced secretionand gene regulation of the potent leukocyte chemoattractant IL-8,generated by intestinal epithelial cells (Gewirtz, A. T., McCormick, B.,Neish, A. S., Petasis, N. A., Gronert, K., Serhan, C. N. & Madara, J. L.(1998) J. Clin. Invest. 101, 1860-1869). It is therefore likely that, inaddition to the inhibition of prostaglandin formation, the benefits ofASA therapy also result from the triggering of novel anti-inflammatorylipid mediators that act locally to down regulate leukocytes.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to compounds having theformulae (I-V):

-   -   wherein X is R₁, OR₁, or SR₁;    -   wherein R₁ is        -   (i) a hydrogen atom;        -   (ii) an alkyl of 1 to 8 carbons atoms, inclusive, which may            be straight chain or branched;        -   (iii) a cycloalkyl of 3 to 10 carbon atoms;        -   (iv) an aralkyl of 7 to 12 carbon atoms;        -   (v) phenyl;        -   (vi) substituted phenyl

-   -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl;        -   (vii) a detectable label molecule; or        -   (viii) a straight or branched chain alkenyl of 2 to 8 carbon            atoms, inclusive;    -   wherein Q₁ is (C═O), SO₂ or (CN), provided when Q₁ is CN, then X        is absent;    -   wherein Q₃ and Q₄ are each independently O, S or NH;    -   wherein one of R₂ and R₃ is a hydrogen atom and the other is        -   (a) H;        -   (b) an alkyl of 1 to 8 carbon atoms, inclusive, which may be            a straight chain or branched;        -   (c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;        -   (d) an alkenyl of 2 to 8 carbon atoms, inclusive, which may            be straight chain or branched; or        -   (e) R_(a)Q₂R_(b) wherein Q₂ is —O— or —S—; wherein R_(a) is            alkylene of 0 to 6 carbons atoms, inclusive, which may be            straight chain or branched and wherein R_(b) is alkyl of 0            to 8 carbon atoms, inclusive, which may be straight chain or            branched, provided when R_(b) is 0, then R_(b) is a hydrogen            atom;    -   wherein R₄ is        -   (a) H;        -   (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be            a straight chain or branched;    -   wherein R₅ is

-   -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl or a substituted or unsubstituted,        branched or unbranched alkyl group;    -   wherein Y₁ is —OH, methyl, —SH, an alkyl of 2 to 4 carbon atoms,        inclusive, straight chain or branched, an alkoxy of 1 to 4        carbon atoms, inclusive, or CH_(a)Z_(b) where a+b=3, a=0 to 3,        b=0 to 3 and Z is cyano, nitro or a halogen;    -   wherein R₆ is        -   (a) H;        -   (b) an alkyl from 1 to 4 carbon atoms, inclusive, straight            chain or branched;

wherein T is O or S, and pharmaceutically acceptable salts thereofexcluding 16-phenoxy-LXA₄ and/or 15-epi-16-(para-fluoro)-phenoxy-LXA₄ incertain embodiments.

In preferred embodiments, X is OR₁ wherein R₁ is a hydrogen atom, analkyl group of 1 to 4 carbon atoms or a pharmaceutically acceptablesalt, Q₁ is C═O, R₂ and R₃, if present, are hydrogen atoms, R₄ is ahydrogen atom or methyl, Q3 and Q₄, if present, are both O, R₆, ifpresent, is a hydrogen atom, Y₁, if present, is OH, T is O and R₅ is asubstituted phenyl, e.g.,

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichmay be a straight chain or branched, and hydroxyl. In certainembodiments for R₅, 15-epi-16-para-fluorophenyl, 15-epi-unsubstitutedphenyl, 16-parafluorophenyl or 16-phenyoxy are excluded.

In another aspect, the present invention is directed to an in vivomethod for modulating a disease or condition associated withpolymorphoneutrophil (PMN) inflammation. The method includesadministering to a subject an effective anti-inflammatory amount of apharmaceutical composition including a compound having one of theabove-described formulae.

In another aspect, the invention is directed to a method for modulatinga disease or condition associated with polymorphoneutrophil (PMN)inflammation. The method includes administering to a subject aneffective anti-inflammatory amount of a pharmaceutical compositionincluding a compound having one of the above-described formulae.

In still another aspect, the present invention is directed topharmaceutical compositions including compounds having theabove-described formulae and a pharmaceutically acceptable carrier. Inone embodiment, a preferred compound is

In a preferred embodiment, the pharmaceutical carrier is not a ketone,e.g., acetone.

In yet another aspect, the present invention is directed to a packagedpharmaceutical composition for treating a PMN responsive state in asubject. The packaged pharmaceutical composition includes a containerholding a therapeutically effective amount of at least one lipoxincompound having one of the formulae described above and instructions forusing the lipoxin compound for treating an PMN responsive state in thesubject.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be more fully understood from the following detaileddescription taken in conjunction with the accompanying drawings, inwhich:

FIG. 1 depicts (A) Initial metabolic step of LXA₄ inactivation in mousewhole blood and 15-oxo-LXA₄ MS/MS spectrum. LXA₄ (21 mM) was incubatedex vivo in mouse whole blood for 3 h. The MS/MS spectrum of the majoroxo-product is indicative of 15-oxo-LXA₄, with diagnostic product ionsat m/z: 349 (a=[M-H]⁻), 331 (a-H₂O), 313 (a-2H₂O), 305 (b=[M-H]⁻-CO₂),287 (b-H₂O), 269 (b-2H₂O), 233 (c), and 217 (c-O). (B) Biostability ofLXA₄ and stable analogs in mouse whole blood. LXA₄, 15R/S)-methyl-LXA₄(ATLa₁, which carries a racemic methyl group at C-15), and15-epi-16-(para-fluoro)-phenoxy-LXA₄ (ATLa₂, in which a bulky(para-fluoro)-phenoxy group replaces the w-chain at C-16) were added(see Methods) to heparinized mouse whole blood and incubated at 37° C.for 0 and 3 h. Following centrifugation at 800×g and 0° C., the plasmasupernatants were drawn off and stopped in two volumes of ice coldmethanol. The lipoxins were extracted by solid phase methodology andquantitated by LC/MS/MS. Values represent means±SEM (n=3−4).

FIG. 2 demonstrates that ATLa₂ inhibits TNF-a-induced PMN infiltrationby both local air pouch and i.v. delivery. When injected locally intothe air pouch, following injection of vehicle (900 ml PBS), murine TNF-a(20 ng/100 ml PBS) induced the infiltration of 4.8±1.1×10⁶ PMN by 4 h.Dexamethasone (10 mg/air pouch), ASA (1 mg/air pouch), and ATLa₂ (10mg/air pouch) were locally administered in 900 ml PBS and prior toTNF-a. Systemic delivery of ATLa₂ was by i.v. injection into the mousetail vein (10 mg/mouse). 2.1±0.7×10⁵ PMN were found in the air pouch 4 hafter injection of vehicle (1 ml sterile PBS) alone. Values representmean±SEM (n=3−5). *P<0.05, †P<0.15 Student's two-tailed t-test

FIG. 3 are representative tissue biopsies of air pouch linings:inhibition of TNF-a-induced PMN accumulation. (A) Lining section taken 4h after exposure to TNF-a (20 ng/mouse) showing increased neutrophilnumber, low-power field inset. (B) Section taken 4 h following exposureto TNF-a (20 ng/mouse), with prior local delivery of ATLa₂ (10mg/mouse). (C) Section taken 4 h following exposure to TNF-a (20ng/mouse), with prior i.v. delivery of ATLa₂ (10 mg/mouse). (D) Sectionof 6-day air pouch lower lining taken from a mouse 4 h followingexposure to vehicle alone. Arrows denote neutrophils. Sections wereprepared as in Methods and stained with hematoxylin-eosin.

FIG. 4 demonstrates that ATLa₂ does not inhibit PMN recruitment to asite of inflammation by regulating vasodilatation. Mouse arterialpressure was monitored with a pressure transducer via the cannulatedcarotid artery. Tail vein injection of vehicle (100 ml; 0.9% saline)showed no changes in arterial pressure while 10 mg Iloprost elicited amaximum mean decrease of ˜28 mmHg˜50 s post-injection, with pressurereturning to baseline after ˜500 s. 10 mg of ATLa₂ were injected into 3mice with no change in mean arterial pressure. Values represent mean±SEM(n=3).

FIG. 5 demonstrates that ATLa₂ inhibits both PMA- and LTB₄-induced PMNinfiltration by topical application and not i.v. injection. ATLa₂ wasapplied topically (20 mg in 10 ml acetone) to the left mouse ear ordelivered intravenously (10 mg in 100 ml of 0.9% sterile saline) throughthe tail vein. Inflammation was induced in left and right ears bytopical application of either LTB₄ (1 mg) or PMA (100 ng) in acetone (10ml). Punch biopsies were obtained after 24 h and MPO activity wasmeasured as an index of PMN number in the ear. Values represent mean±SEM(n=3). *P<0.05 Student's two-tailed t-test

FIG. 6 depicts ATLa₂ bolus tail vein injection: time course in plasma.BALB/c mice (6-8 wk) received i.v. tail vein injections of ATLa₂ (2mg/mouse) in 100 ml sterile 0.9% saline. Blood was obtained by cardiacpuncture and ATLa₂ was extracted from the plasma by solid phaseextraction. The amounts of ATLa₂ remaining were quantitated by LC/MS/MS.Values represent mean±SEM (n=3).

FIG. 7 represents methyl ester hydrolysis of ATLa₂ to free acid in exvivo mouse whole blood. ATLa₂ was incubated ex vivo in mouse whole blood(2.8 μM) for 0, 2, 5, 10, 15, or 180 minutes. The incubations werestopped by chilling the blood on ice for one minute followed bycentrifugation (800×g) at 0° C. The plasma supernatant was stopped in 2vol of ice cold methanol. The samples were prepared for analysis bysolid phase extraction and the amount of free acid in the sample wasquantitated by LC/MS/MS. Within 10-15 minutes, 100% of the ATLa₂ methylester is hydrolyzed to the free acid without loss of compound. Valuesrepresent mean±SEM (n=3).

FIG. 8 demonstrates that ATL analogs induce vasodilation in isolated rataorta: relaxation relative to Iloprost. Rats were euthanized withpentobarbital overdoses. The aorta was isolated and a pre-load of300-400 mg given. The vessels were pre-contracted with U46619 (25ng/ml). Relaxation was induced with addition of Iloprost,5(R/S)-methyl-LXB₄, ATLa₁, or ATLa₂ to a final concentration of μM.Aorta smooth muscle relaxation was measured with a force transducer andthe data digitized and stored on a PC. 5(R/S)-methyl-LXB₄, ATLa₁, andATLa₂ caused 42.8%, 37.0%, 38.1%, and 40.0% relaxation of smooth muscle,respectively. Values represent ±SEM (n=4−6).

DETAILED DESCRIPTION OF THE INVENTION

The features and other details of the invention will now be moreparticularly described and pointed out in the claims. It will beunderstood that the particular embodiments of the invention are shown byway of illustration and not as limitations of the invention. Theprinciple features of this invention can be employed in variousembodiments without departing from the scope of the invention.

Aspirin (ASA) triggers a switch in the biosynthesis of lipid mediators,inhibiting prostanoid production and initiating 15-epi-lipoxingeneration, through the acetylation of cyclooxygenase II. Theseaspirin-triggered lipoxins (ATL) may mediate some of ASA's beneficialactions and therefore are of interest in the search for novelanti-inflammatories that could manifest fewer unwanted side-effects.Design modifications to native ATL structure prolong its biostability invivo. In mouse whole blood, ATL analogs protected at carbon 15 (ATLa₁)and the omega end (ATLa₂) were recoverable to ˜90 and 100% at 3 hours,respectively, compared to a ˜40% loss of native lipoxin A₄ (LXA₄). ATLa₂retains bioactivity and, at levels as low as ˜24 nmol/mouse, potentlyinhibited TNF-a-induced leukocyte recruitment into the dorsal air pouch.Inhibition was evident by either local intra-air pouch delivery (˜77%inhibition) or via systemic delivery by intravenous injection (˜85%inhibition) and proved more potent than local delivery of either ASA ordexamethasone. Rank order for inhibiting PMN infiltration was: ATLa₂ (10mg, i.v.)≈ATLa₂ (10 mg, local)>ASA (1.0 mg, local) dexamethasone (10 mg,local). Applied topically to mouse ear skin, ATLa₂ also inhibited PMNinfiltration induced by leukotriene B₄ (˜78% inhibition) or phorbolester, which initiates endogenous chemokine production (˜49%inhibition). These results indicate that this fluorinated analog ofnatural aspirin-triggered LXA₄ is bioavailable by either local orsystemic delivery routes and is a more potent and precise inhibitor ofneutrophil accumulation than ASA.

Abbreviations: ASA, Aspirin, acetylsalicylic acid; ATL,aspirin-triggered lipoxins; ATLa₁, 15(R/S)-methyl-lipoxin A₄; ATLa₂,15-epi-16-(para-fluoro)-phenoxy-lipoxin A₄; COX I and II,cyclooxygenases I and II; 15-epi-LXA₄, 15-epi-lipoxin A₄,5S,6R,15R-trihydroxyeicosa-7E,9E,11Z,13E-tetraenoic acid; 15-epi-LXB₄,15-epi-lipoxin B₄, 5S,14R,15R-trihydroxyeicosa-6E,8Z,10E,12E-tetraenoicacid; i.v., intravenous; LC/MS/MS, liquid chromatography-tandem massspectrometry; LTB₄, leukotriene B₄,5S,12R-dihydroxyeicosa-6E,8Z,10Z,14E-eicosatetraenoic acid; LX,lipoxins; LXA₄, lipoxin A₄,5S,6R,15S-trihydroxyeicosa-7E,9E,11Z,13E-tetraenoic acid; LXB₄, lipoxinB₄, 5S,14R,15S-trihydroxyeicosa-6E,8Z,10E,12E-tetraenoic acid; PG,prostaglandin; PMA, phorbol 12-myristate 13-acetate; PMN,polymorphonuclear leukocyte; TNF-a, tumor necrosis factor a.

PMN accumulation and activation play central roles in the pathogenesisof a wide range of disease states as diverse as rheumatoid arthritis,atherosclerosis, ulcerative colitis, and psoriasis (Pillinger, M. H. &Abramson, S. B. (1995) Rheum. Dis. Clin. North Am. 21, 691-714;Hagihara, H., Nomoto, A., Mutoh, S., Yamaguchi, I. & Ono, T. (1991)Atherosclerosis 91, 107-116; McLaughlan, J. M., Seth, R., Vautier, G.,Robins, R. A., Scott, B. B., Hawkey, C. J. & Jenkins, D. (1997) J.Pathol. 181, 87-92; Anezaki, K., Asakura, H., Honma, T., Ishizuka, K.,Funakoshi, K., Tsukada, Y. & Narisawa, R. (1998) Intern. Med. 37,253-258; Iverson, L. & Kragballe, K. (1997) in Skin Immune System (SIS),ed. Bos, J. D. (CRC Press, Boca Raton), pp. 227-237). Hence theelucidation of endogenous regulatory mechanisms that can controlneutrophil functions are of considerable therapeutic interest. Becausethey are small lipophilic compounds amenable to total organic synthesis,the natural lipoxins, and specifically their endogenous isoform ATL, arewell suited as potential leads for novel small molecule therapeutics aswell as pharmacologic tools for uncovering endogenous counter-regulatoryand/or anti-inflammatory signaling pathways.

Design modifications that enhance biostability are advantageous sincethe lipoxins are autacoids that are rapidly biosynthesized in responseto stimuli, in turn elicit counter-regulatory responses, and then arerapidly enzymatically inactivated (Serhan, C. N. (1997) Prostaglandins53, 107-137). 15-Hydroxy-prostaglandin dehydrogenase (15-PGDH), whichcatalyzes the reversible oxidation of the carbon-15 position alcoholgroup of prostaglandins and several other w-6-hydroxylated fatty acids,also catalyzes the first step of lipoxin inactivation (FIG. 1A) (Ensor,C. M. & Tai, H.-H. (1991) in Prostaglandins, Leukotrienes, Lipoxins, andPAF, ed. Bailey, J. M. (Plenum Press, New York), pp. 39-52; Serhan, C.N., Fiore, S., Brezinski, D. A. & Lynch, S. (1993) Biochemistry 32,6313-6319; Maddox, J. F., Colgan, S. P., Clish, C. B., Petasis, N. A.,Fokin, V. V. & Serhan, C. N. (1998) FASEB J. 12, 487-494). In view ofthese findings, several stable analogs of ATL and LXA₄ were designedthat resist oxidation at carbon-15 by recombinant dehydrogenase in vitro(Serhan, C. N., Maddox, J. F., Petasis, N. A., Akritopoulou-Zanze, I.,Papayianni, A., Brady, H. R., Colgan, S. P. & Madara, J. L. (1995)Biochemistry 34, 14609-14615). These LX act at LXA₄ receptors onleukocytes and are active within the nanomolar range: inhibiting PMNadherence, transmigration, and diapedesis (Takano, T., Clish, C. B.,Gronert, K., Petasis, N. & Serhan, C. N. (1998) J. Clin. Invest. 101,819-826). Design modifications to native ATL biostabilize thesemediators in whole blood to resist rapid inactivation. Moreover, thefluorinated ATL analog, namely 15-epi-16-(para-fluoro)-phenoxy-LXA₄(ATLa₂), is a potent inhibitor of PMN recruitment in murine in vivomodels when administered through both local and systemic routes.

The present invention is directed to new lipoxin compounds. In oneembodiment, the compound has the formula (I)

-   -   wherein X is R₁, OR₁, or SR₁;    -   wherein R₁ is        -   (i) a hydrogen atom;        -   (ii) an alkyl of 1 to 8 carbons atoms, inclusive, which may            be straight chain or branched;        -   (iii) a cycloalkyl of 3 to 10 carbon atoms;        -   (iv) an aralkyl of 7 to 12 carbon atoms;        -   (v) phenyl;        -   (vi) substituted phenyl

-   -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl;        -   (vii) a detectable label molecule; or        -   (viii) a straight or branched chain alkenyl of 2 to 8 carbon            atoms, inclusive;    -   wherein Q₁ is (C═O), SO₂ or (CN), provided when Q₁ is CN, then X        is absent;    -   wherein Q₃ and Q₄ are each independently O, S or NH;    -   wherein one of R₂ and R₃ is a hydrogen atom and the other is        -   (a) H;        -   (b) an alkyl of 1 to 8 carbon atoms, inclusive, which may be            a straight chain or branched;        -   (c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;        -   (d) an alkenyl of 2 to 8 carbon atoms, inclusive, which may            be straight chain or branched; or        -   (e) R_(a)Q₂R_(b) wherein Q₂ is —O— or —S—; wherein R_(a) is            alkylene of 0 to 6 carbons atoms, inclusive, which may be            straight chain or branched and wherein R_(b) is alkyl of 0            to 8 carbon atoms, inclusive, which may be straight chain or            branched, provided when R_(b) is 0, then R_(b) is a hydrogen            atom;    -   wherein R₄ is        -   (a) H;        -   (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be            a straight chain or branched;    -   wherein R₅ is

-   -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl or a substituted or unsubstituted,        branched or unbranched alkyl group;    -   wherein Y₁ is —OH, methyl, —SH, an alkyl of 2 to 4 carbon atoms,        inclusive, straight chain or branched, an alkoxy of 1 to 4        carbon atoms, inclusive, or CH_(a)Z_(b) where a+b=3, a═0 to 3,        b═0 to 3 and Z is cyano, nitro or a halogen;    -   wherein R₆ is        -   (a) H;        -   (b) an alkyl from 1 to 4 carbon atoms, inclusive, straight            chain or branched;

wherein T is O or S, and pharmaceutically acceptable salts thereof,excluding 16-phenoxy-LXA₄ and 15-epi-16-(para-fluoro)-phenoxy-LXA₄.

In another embodiment, compounds useful in the invention have theformula (II)

-   -   wherein X is R₁, OR₁, or SR₁;    -   wherein R₁ is        -   (i) a hydrogen atom;        -   (ii) an alkyl of 1 to 8 carbons atoms, inclusive, which may            be straight chain or branched;        -   (iii) a cycloalkyl of 3 to 10 carbon atoms;        -   (iv) an aralkyl of 7 to 12 carbon atoms;        -   (v) phenyl;        -   (vi) substituted phenyl

-   -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl;        -   (vii) a detectable label molecule; or        -   (viii) a straight or branched chain alkenyl of 2 to 8 carbon            atoms, inclusive;    -   wherein Q₁ is (C═O), SO₂ or (CN), provided when Q₁ is CN, then X        is absent;    -   wherein one of R₂ and R₃ is a hydrogen atom and the other is        -   (a) H;        -   (b) an alkyl of 1 to 8 carbon atoms, inclusive, which may be            a straight chain or branched;        -   (c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;        -   (d) an alkenyl of 2 to 8 carbon atoms, inclusive, which may            be straight chain or branched; or        -   (e) R_(a)Q₂R_(b) wherein Q₂ is —O— or —S—; wherein R_(a) is            alkylene of 0 to 6 carbons atoms, inclusive, which may be            straight chain or branched and wherein R_(b) is alkyl of 0            to 8 carbon atoms, inclusive, which may be straight chain or            branched, provided when R_(b) is 0, then R_(b) is a hydrogen            atom;    -   wherein R₄ is        -   (a) H;        -   (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be            a straight chain or branched;    -   wherein R₅ is

-   -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl or a substituted or unsubstituted,        branched or unbranched alkyl group;    -   wherein Y₁ is —OH, methyl, —SH, an alkyl of 2 to 4 carbon atoms,        inclusive, straight chain or branched, an alkoxy of 1 to 4        carbon atoms, inclusive, or CH_(a)Z_(b) where a+b=3, a=0 to 3,        b═0 to 3 and Z is cyano, nitro or a halogen;    -   wherein R₆ is        -   (a) H;        -   (b) an alkyl from 1 to 4 carbon atoms, inclusive, straight            chain or branched;

wherein T is O or S, and pharmaceutically acceptable salts thereof,excluding 16-phenoxy-LXA₄ and 15-epi-16-(para-fluoro)-phenoxy-LXA₄.

The invention is also directed to useful lipoxin compounds having theformula (III)

-   -   wherein X is R₁, OR₁, or SR₁;    -   wherein R₁ is        -   (i) a hydrogen atom;        -   (ii) an alkyl of 1 to 8 carbons atoms, inclusive, which may            be straight chain or branched;        -   (iii) a cycloalkyl of 3 to 10 carbon atoms;        -   (iv) an aralkyl of 7 to 12 carbon atoms;        -   (v) phenyl;        -   (vi) substituted phenyl

-   -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl;        -   (vii) a detectable label molecule; or        -   (viii) a straight or branched chain alkenyl of 2 to 8 carbon            atoms, inclusive;    -   wherein Q₁ is (C═O), SO₂ or (CN), provided when Q₁ is CN, then X        is absent;    -   wherein one of R₂ and R₃ is a hydrogen atom and the other is        -   (a) H;        -   (b) an alkyl of 1 to 8 carbon atoms, inclusive, which may be            a straight chain or branched;        -   (c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;        -   (d) an alkenyl of 2 to 8 carbon atoms, inclusive, which may            be straight chain or branched; or        -   (e) R_(a)Q₂R_(b) wherein Q₂ is —O— or —S—; wherein R_(a) is            alkylene of 0 to 6 carbons atoms, inclusive, which may be            straight chain or branched and wherein R_(b) is alkyl of 0            to 8 carbon atoms, inclusive, which may be straight chain or            branched, provided when R_(b) is 0, then R_(b) is a hydrogen            atom;    -   wherein R₄ is        -   (a) H;        -   (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be            a straight chain or branched;    -   wherein R₅ is

-   -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl or a substituted or unsubstituted,        branched or unbranched alkyl group;    -   wherein R₆ is        -   (a) H;        -   (b) an alkyl from 1 to 4 carbon atoms, inclusive, straight            chain or branched;

wherein T is O or S, and pharmaceutically acceptable salts thereof,excluding 16-phenoxy-LXA₄ and 15-epi-16-(para-fluoro)-phenoxy-LXA₄.

The invention is further directed to useful lipoxin compounds having theformula (IV)

-   -   wherein X is R₁, OR₁, or SR₁;    -   wherein R₁ is        -   (i) a hydrogen atom;        -   (ii) an alkyl of 1 to 8 carbons atoms, inclusive, which may            be straight chain or branched;        -   (iii) a cycloalkyl of 3 to 10 carbon atoms;        -   (iv) an aralkyl of 7 to 12 carbon atoms;        -   (v) phenyl;        -   (vi) substituted phenyl

-   -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl;        -   (vii) a detectable label molecule; or        -   (viii) a straight or branched chain alkenyl of 2 to 8 carbon            atoms, inclusive;    -   wherein Q₁ is (C═O), SO₂ or (CN), provided when Q₁ is CN, then X        is absent;    -   wherein R₄ is        -   (a) H;        -   (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be            a straight chain or branched;    -   wherein R₅ is

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichmay be a straight chain or branched, and hydroxyl or a substituted orunsubstituted, branched or unbranched alkyl group;

-   -   wherein R₆ is        -   (a) H;        -   (b) an alkyl from 1 to 4 carbon atoms, inclusive, straight            chain or branched;

wherein T is O or S, and pharmaceutically acceptable salts thereof,excluding 16-phenoxy-LXA₄ and 15-epi-16-(para-fluoro)-phenoxy-LXA₄.

The invention is further directed to useful lipoxin compounds having theformula (V)

-   -   wherein X is R₁, OR₁, or SR₁;    -   wherein R₁ is        -   (i) a hydrogen atom;        -   (ii) an alkyl of 1 to 8 carbons atoms, inclusive, which may            be straight chain or branched;        -   (iii) a cycloalkyl of 3 to 10 carbon atoms;        -   (iv) an aralkyl of 7 to 12 carbon atoms;        -   (v) phenyl;        -   (vi) substituted phenyl

-   -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl;        -   (vii) a detectable label molecule; or        -   (viii) a straight or branched chain alkenyl of 2 to 8 carbon            atoms, inclusive;    -   wherein Q₁ is (C═O), SO₂ or (CN), provided when Q₁ is CN, then X        is absent;    -   wherein R₄ is        -   (a) H;        -   (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be            a straight chain or branched;    -   wherein R₅ is

-   -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl or a substituted or unsubstituted,        branched or unbranched alkyl group;    -   wherein R₆ is        -   (a) H;        -   (b) an alkyl from 1 to 4 carbon atoms, inclusive, straight            chain or branched; and

pharmaceutically acceptable salts thereof, excluding 16-phenoxy-LXA₄ and15-epi-16-(para-fluoro)-phenoxy-LXA₄.

In preferred embodiments, X is OR₁ wherein R₁ is a hydrogen atom, analkyl group of 1 to 4 carbon atoms or a pharmaceutically acceptablesalt, Q₁ is C═O, R₂ and R₃, if present, are hydrogen atoms, R₄ is ahydrogen atom or methyl, Q₃ and Q₄, if present, are both O, R₆, ifpresent, is a hydrogen atom, Y₁, if present, is OH, T is O and R₅ is asubstituted phenyl, e.g.,

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichmay be a straight chain or branched, and hydroxyl or a substituted orunsubstituted, branched or unbranched alkyl group. In certainembodiments para-fluorophenyl and unsubstituted phenyl groups areexcluded from R₅.

In another aspect, the present invention is directed to an in vivomethod for modulating a disease or condition associated withpolymorphoneutrophil (PMN) inflammation. The method includesadministering to a subject an effective anti-inflammatory amount of apharmaceutical composition including a compound having one of theabove-described formulae.

In another aspect, the invention is directed to a method for modulatinga disease or condition associated with polymorphoneutrophil (PMN)inflammation. The method includes administering to a subject aneffective anti-inflammatory amount of a pharmaceutical compositionincluding a compound having one of the above-described formulae.

In still another aspect, the present invention is directed topharmaceutical compositions including compounds having theabove-described formulae and a pharmaceutically acceptable carrier. Inone embodiment, a preferred compound is

In a preferred embodiment, the pharmaceutical carrier is not a ketone,e.g., acetone.

In one embodiment, the antiinflammatories of the invention can beincorporated into a shampoo or a body cleansing product, e.g., a soap,for cleansing of the scalp and/or body. The use of these compounds in ashampoo or soap product can be used to treat psoriasis, seborrheicdermatitis, pustular dermatosis and dandruff. Thus the compounds areuseful for modulating PMN inflammation associated with such conditions.

In yet another aspect, the present invention is directed to a packagedpharmaceutical composition for treating a PMN responsive state in asubject. The packaged pharmaceutical composition includes a containerholding a therapeutically effective amount of at least one lipoxincompound having one of the formulae described above and instructions forusing the lipoxin compound for treating an PMN responsive state in thesubject.

In preferred embodiments, Y₁ is a hydroxyl and the carbon bearing thehydroxyl can have an R or S configuration. In most preferredembodiments, the chiral carbon bearing the hydroxyl group, e.g., Y₁ isdesignated as a 15-epi-lipoxin as is known in the art.

In certain embodiments the chirality of the carbons bearing the R₂, R₃,Q₃ and Q₄ groups can each independently be either R or S. In preferredembodiments, Q₃ and Q₄ have the chiralities shown in structures II, III,IV or V.

In preferred embodiments, R₄ is a hydrogen. In other preferredembodiments, R₆ is a hydrogen.

Additionally, R₅ can be a substituted or unsubstituted, branched orunbranched alkyl group having between 1 and about 6 carbon atoms,preferably between 1 and 4 carbon atoms, most preferably between 1 and3, and preferably one or two carbon atoms. The carbon atoms can havesubstituents which include halogen atoms, hydroxyl groups, or ethergroups.

The compounds encompassed by U.S. Pat. No. 5,441,951 are excluded fromcertain aspects of the present invention.

The compounds useful in the present invention can be prepared by thefollowing synthetic scheme:

wherein X, Q₁, Q₃, Q₄, R₂, R₃, R₄, R₅, R₆, Y₁ and T are as definedabove. Suitable methods known in the art to can be used to produce eachfragment. For example, the acetylenic fragment can be prepared by themethods discussed in Nicolaou, K. C. et al. (1991) Angew. Chem. Int. Ed.Engl. 30:1100; Nicolaou, K. C. et al. (1989) J. Org. Chem. 54:5527;Webber, S. E. et al. (1988) Adv. Exp. Med. Biol. 229:61; and U.S. Pat.No. 5,441,951. The second fragment can be prepared by the methods ofRaduchel, B. and Vorbruggen, H. (1985) Adv. Prostaglandin ThromboxaneLeukotriene Res. 14:263.

A “lipoxin analog” shall mean a compound which has an “active region”that functions like the active region of a “natural lipoxin”, but whichhas a “metabolic transformation region” that differs from naturallipoxin. Lipoxin analogs include compounds which are structurallysimilar to a natural lipoxin, compounds which share the same receptorrecognition site, compounds which share the same or similar lipoxinmetabolic transformation region as lipoxin, and compounds which areart-recognized as being analogs of lipoxin. Lipoxin analogs includelipoxin analog metabolites. The compounds disclosed herein may containone or more centers of asymmetry. Where asymmetric carbon atoms arepresent, more than one stereoisomer is possible, and all possibleisomeric forms are intended to be included within the structuralrepresentations shown. Optically active (R) and (S) isomers may beresolved using conventional techniques known to the ordinarily skilledartisan. The present invention is intended to include the possiblediastereiomers as well as the racemic and optically resolved isomers.

The terms “corresponding lipoxin” and “natural lipoxin” refer to anaturally-occurring lipoxin or lipoxin metabolite. Where an analog hasactivity for a lipoxin-specific receptor, the corresponding or naturallipoxin is the normal ligand for that receptor. For example, where ananalog is a LXA₄ specific receptor on differentiated HL-60 cells, thecorresponding lipoxin is LXA₄. Where an analog has activity as anantagonist to another compound (such as a leukotriene), which isantagonized by a naturally-occurring lipoxin, that natural lipoxin isthe corresponding lipoxin.

“Active region” shall mean the region of a natural lipoxin or lipoxinanalog, which is associated with in vivo cellular interactions. Theactive region may bind the “recognition site” of a cellular lipoxinreceptor or a macromolecule or complex of macromolecules, including anenzyme and its cofactor. Preferred lipoxin A₄ analogs have an activeregion comprising C₅-C₁₅ of natural lipoxin A₄. Preferred lipoxin B₄analogs have an active region comprising C5-C14 of natural lipoxin B4.

The term “recognition site” or receptor is art-recognized and isintended to refer generally to a functional macromolecule or complex ofmacromolecules with which certain groups of cellular messengers, such ashormones, leukotrienes, and lipoxins, must first interact before thebiochemical and physiological responses to those messengers areinitiated. As used in this application, a receptor may be isolated, onan intact or permeabilized cell, or in tissue, including an organ. Areceptor may be from or in a living subject, or it may be cloned. Areceptor may normally exist or it may be induced by a disease state, byan injury, or by artificial means. A compound of this invention may bindreversibly, irreversibly, competitively, noncompetitively, oruncompetitively with respect to the natural substrate of a recognitionsite.

The term “metabolic transformation region” is intended to refergenerally to that portion of a lipoxin, a lipoxin metabolite, or lipoxinanalog including a lipoxin analog metabolite, upon which an enzyme or anenzyme and its cofactor attempts to perform one or more metabolictransformations which that enzyme or enzyme and cofactor normallytransform on lipoxins. The metabolic transformation region may or maynot be susceptible to the transformation. A nonlimiting example of ametabolic transformation region of a lipoxin is a portion of LXA₄ thatincludes the C-13,14 double bond or the C-15 hydroxyl group, or both.

The term “detectable label molecule” is meant to include fluorescent,phosphorescent, and radiolabeled molecules used to trace, track, oridentify the compound or receptor recognition site to which thedetectable label molecule is bound. The label molecule may be detectedby any of the several methods known in the art.

The term “labeled lipoxin analog” is further understood to encompasscompounds which are labeled with radioactive isotopes, such as but notlimited to tritium (³H), deuterium (²H), carbon (¹⁴C), or otherwiselabeled (e.g. fluorescently). The compounds of this invention may belabeled or derivatized, for example, for kinetic binding experiments,for further elucidating metabolic pathways and enzymatic mechanisms, orfor characterization by methods known in the art of analyticalchemistry.

The term “inhibits metabolism” means the blocking or reduction ofactivity of an enzyme which metabolizes a native lipoxin. The blockageor reduction may occur by covalent bonding, by irreversible binding, byreversible binding which has a practical effect of irreversible binding,or by any other means which prevents the enzyme from operating in itsusual manner on another lipoxin analog, including a lipoxin analogmetabolite, a lipoxin, or a lipoxin metabolite.

The term “resists metabolism” is meant to include failing to undergo oneor more of the metabolic degradative transformations by at least one ofthe enzymes which metabolize lipoxins. Two nonlimiting examples of LXA₄analog that resists metabolism are 1) a structure which can not beoxidized to the 15-oxo form, and 2) a structure which may be oxidized tothe 15-oxo form, but is not susceptible to enzymatic reduction to the13,14-dihydro form.

The term “more slowly undergoes metabolism” means having slower reactionkinetics, or requiring more time for the completion of the series ofmetabolic transformations by one or more of the enzymes which metabolizelipoxin. A nonlimiting example of a LXA₄ analog which more slowlyundergoes metabolism is a structure which has a higher transition stateenergy for C-15 dehydrogenation than does LXA₄ because the analog issterically hindered at the C-16.

The term “tissue” is intended to include intact cells, blood, bloodpreparations such as plasma and serum, bones, joints, muscles, smoothmuscles, and organs.

The term “halogen” is meant to include fluorine, chlorine, bromine andiodine, or fluoro, chloro, bromo, and iodo. In certain aspects, thecompounds of the invention do not include halogenated compounds, e.g.,fluorinated compounds.

The term “subject” is intended to include living organisms susceptibleto conditions or diseases caused or contributed to by inflammation,inflammatory responses, vasoconstriction, and myeloid suppression.Examples of subjects include humans, dogs, cats, cows, goats, and mice.The term subject is further intended to include transgenic species.

When the compounds of the present invention are administered aspharmaceuticals, to humans and mammals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting a compound(s) of thepresent invention within or to the subject such that it can perform itsintended function. Typically, such compounds are carried or transportedfrom one organ, or portion of the body, to another organ, or portion ofthe body. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;phosphate buffer solutions; and other non-toxic compatible substancesemployed in pharmaceutical formulations.

In certain embodiment, the compounds of the present invention maycontain one or more acidic functional groups and, thus, are capable offorming pharmaceutically acceptable salts with pharmaceuticallyacceptable bases. The term “pharmaceutically acceptable salts” in theseinstances refers to the relatively non-toxic, inorganic and organic baseaddition salts of compounds of the present invention. These salts canlikewise be prepared in situ during the final isolation and purificationof the compounds, or by separately reacting the purified compound in itsfree acid form with a suitable base, such as the hydroxide, carbonate orbicarbonate of a pharmaceutically acceptable metal cation, with ammonia,or with a pharmaceutically acceptable organic primary, secondary ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum saltsand the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like.

The term “pharmaceutically acceptable esters” refers to the relativelynon-toxic, esterified products of the compounds of the presentinvention. These esters can be prepared in situ during the finalisolation and purification of the compounds, or by separately reactingthe purified compound in its free acid form or hydroxyl with a suitableesterifying agent. Carboxylic acids can be converted into esters viatreatment with an alcohol in the presence of a catalyst. The term isfurther intended to include lower hydrocarbon groups capable of beingsolvated under physiological conditions, e.g., alkyl esters, methyl,ethyl and propyl esters. In a preferred embodiment, the ester is not amethyl ester (See, for example, Berge et al., supra.).

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable forintravenous, oral, nasal, topical, transdermal, buccal, sublingual,rectal, vaginal and/or parenteral administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods well known in the art of pharmacy. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compound whichproduces a therapeutic effect. Generally, out of one hundred per cent,this amount will range from about 1 percent to about ninety-nine percentof active ingredient, preferably from about 5 percent to about 70percent, most preferably from about 10 percent to about 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; solutionretarding agents, such as paraffin; absorption accelerators, such asquaternary ammonium compounds; wetting agents, such as, for example,cetyl alcohol and glycerol monostearate; absorbents, such as kaolin andbentonite clay; lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and coloring agents. In the case of capsules, tabletsand pills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the activecompound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given by formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Intravenous injection administration is preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrastemal injection and infusion.

The phrases “systemic administration,” “administered systematically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of ordinary skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, intravenous andsubcutaneous doses of the compounds of this invention for a patient,when used for the indicated analgesic effects, will range from about0.0001 to about 100 mg per kilogram of body weight per day, morepreferably from about 0.01 to about 50 mg per kg per day, and still morepreferably from about 0.1 to about 40 mg per kg per day. For example,between about 0.01 microgram and 20 micrograms, between about 20micrograms and 100 micrograms and between about 10 micrograms and 200micrograms of the compounds of the invention are administered per 20grams of subject weight.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical composition.

Materials and Methods

Biostability of LX analogs in mouse whole blood. The analogs ATLa₁ andATLa₂ were prepared by total organic synthesis and their structuresconfirmed by NMR (Serhan, C. N., Maddox, J. F., Petasis, N. A.,Akritopoulou-Zanze, I., Papayianni, A., Brady, H. R., Colgan, S. P. &Madara, J. L. (1995) Biochemistry 34, 14609-14615) (See also U.S. Pat.Nos. 5,441,951, 5,648,512, 5,650,435 and 5,750,354, incorporated hereinby reference, for suitable examples of syntheses). Male BALB/c mice (6-8wk) (Harlan Sprague Dawley, Inc.) were anesthetized with pentobarbital(70 mg/kg) and whole blood was drawn via cardiac puncture into heparin(500 U/ml). LXA₄, ATLa₁, and ATLa₂ (2.4 mM) were incubated in 250 ml ofblood (37° C.) for either 0 or 3 h. For time zero (T=0), the bloodaliquots were placed in an ice bath for 1 min and, immediately after theaddition of LXA₄ or ATLa, were centrifuged at 800×g at 0° C. for 20 min.The plasma supernatants were collected, stopped in 400 ml of ice coldmethanol, and stored at −20° C. prior to solid phase extraction. For T=3h, the blood aliquots were incubated with ATLa and gently mixed byshaking at 37° C. After each incubation period, the plasma was collectedand stopped as above. Prostaglandin B₂ (Oxford Biomedical Research,Inc., Oxford, Mich.) was added to the blood samples immediately beforecentrifugation as an internal standard for extraction recovery.Denatured protein precipitates were pelleted from the stopped plasmasamples and were washed twice with 200 ml of methanol. The plasmasupernatant and washes were pooled and extracted with Extract-Cleansolid phase extraction cartridges (500 mg C₁₈, Alltech Associates Inc.,Deerfield, Ill.). The methyl formate fractions were taken to drynesswith a gentle stream of nitrogen and suspended in methanol for injectionand quantitative analyses by UV spectrophotometry and LC/MS/MS.

LC/MS/MS analyses. LC/MS/MS was performed employing an LCQ (FinniganMAT, San Jose, Calif.) quadrupole ion trap mass spectrometer systemequipped with an electrospray atmospheric pressure ionization probe.Samples were suspended in methanol and injected into the HPLC component,which consisted of a SpectraSYSTEM P4000 (Thermo Separation Products,San Jose, Calif.) quaternary gradient pump, a Prodigy octadecylsilane-3(100×2 mm, 5 mm) column (Phenomenex, Torrance, Calif.) or a LUNA C18-2(150×2 mm, 5 mm) column, and a rapid spectra scanning SpectraSYSTEMUV2000 (Thermo Separation Products, San Jose, Calif.) UV/VIS absorbancedetector. The column was eluted isocratically with methanol/water/aceticacid (65:35:0.01, v/v/v) at 0.2 ml/min into the electrospray probe. Thespray voltage was set to 5-6 kV and the heated capillary to 250° C. LXA₄and the ATLa were quantitated by selected ion monitoring (SIM) foranalyte molecular anions (e.g. [M-H]⁻=m/z 351.5 for LXA₄, m/z 365.5 forATLa₁, and m/z 405.5 for ATLa₂ free acid) or by UV absorbance at 300 nm.Product ion mass spectra (MS/MS) were also acquired for definitiveidentification of the compounds.

PMN infiltration into mouse air pouch. While male BALB/c mice (6-8 wk)were anesthetized with isoflurane, dorsal air pouches were raised byinjecting 3 ml of sterile air subcutaneously on days 0 and 3 (as inref.) (Sin, Y. M., Sedgwick, A. D., Chea, E. P. & Willoughby, D. A.(1986) Ann. Rheum. Dis. 45, 873-877). On day 6 and while the mice wereanesthetized with isoflurane, 10 mg of ATLa₂ was delivered as a bolusinjection into either the tail vein in 100 ml of sterile 0.9% saline orlocally into the air pouch in 900 ml of PBS −/− (Dulbecco's PhosphateBuffered Saline without magnesium or calcium ions, BioWhittaker,Walkersville, Md.). Dexamethasone and ASA (Sigma Chemical Co., St.Louis, Mo.) were delivered locally as 10 mg and 1.0 mg doses in 900 mlof PBS −/−, respectively. Inflammation in the air pouch was induced bylocal injection of recombinant murine TNF-a (20 ng) (BoehringerMannheim, Indianapolis, Ind.) dissolved in 100 ml of sterile PBS. Whilethe mice were anesthetized with isoflurane, the air pouches were lavagedtwice with 3 ml of sterile PBS 4 h after the initial TNF-a injection.Aspirates were centrifuged at 2000 rpm for 15 min at 23° C. Thesupernatants were removed and the cells were suspended in 500 ml of PBS.Aliquots of the cell suspension were stained with Trypan Blue andenumerated by light microscopy. 50 ml of the resuspended aspirate cellswere added to 150 ml of 30% BSA and centrifuged onto microscope slidesat 2200 rpm for 4 min using a Cytofuge (StatSpin, Norwood, Mass.).Slides were allowed to air dry and were stained with Wright Giemsa stain(Sigma Chemical Co., St. Louis, Mo.) for determination of differentialleukocyte counts. For microscopic analysis, tissues were obtained with a6 mm tissue biopsy punch (Acu-Punch, Acuderm, Inc., Ft. Lauderdale,Fla.) and fixed in 10% buffered formaldehyde. Samples were then embeddedin paraffin, sliced, and stained with hematoxylin-eosin.

Arterial pressure. Male BALB/c mice (6-8 wk, 20 g) were anesthetizedwith pentobarbital (80 mg/kg). The trachea was isolated and a smallpolyethylene catheter (PE50) was introduced to maintain a patent airway.The right carotid artery was isolated and cannulated with PE10 tubingfilled with heparinized (10 units/ml) normal saline. The arterialcatheter was connected to a pressure transducer (World PrecisionInstruments, Sarasota, FA) and the arterial pressure tracing wasrecorded continuously (Astromed MT95K2, West Warwick, R.I.). Allsurgical manipulations were performed using a surgical microscope (CarlZeiss, Inc., Thornwood, N.Y.).

PMN infiltration into ear skin. The mouse ear inflammation model wasused to evaluate the impacts of i.v. and topical deliveries of ATLa₂ onLTB₄- and PMA-induced PMN infiltration (Takano, T., Clish, C. B.,Gronert, K., Petasis, N. & Serhan, C. N. (1998) J. Clin. Invest. 101,819-826). Briefly, ATLa₂ was either applied topically (20 mg in 10 mlacetone) to the inner side of the left mouse ear with vehicle appliedcontralaterally, or delivered as a bolus injection (10 mg in 100 ml of0.9% sterile saline) through the tail vein. 5-7 min later, inflammationwas induced in left and right ears of the mice that received topicalATLa₂ (left ear only in the mice receiving i.v. delivery of ATLa₂) bytopical application of either LTB₄ (1 mg) or PMA (100 ng) in acetone (10ml). After 24 h, 6 mm diameter tissue punch biopsies were taken(Acu-Punch, Acuderm, Inc., Ft. Lauderdale, Fla.) from the ears andassayed by the method of Bradley et al for myeloperoxidase (MPO)activity as an index of PMN number. Isolated murine PMN were enumeratedby light microscopy and processed in the same manner to obtain acalibration curve (Bradley, P. P., Priebat, D. A., Christensen, R. D. &Rothstein, G. (1982) J. Invest. Dermatol. 78,206-209).

Plasma clearance. The time course for the clearance of ATLa₂ from plasmafollowing tail vein injection was determined over 50 min. Male BALB/cmice (6-8 wk, 20 g) were anesthetized with pentobarbital (70 mg/kg) andreceived bolus tail vein injections of 27 mM ATLa₂ (0.1 mg/kg) in 100 mlof sterile 0.9% saline. Blood was taken from the mice by cardiacpuncture at 2, 5, 10, 15, and 50 min post-injection. The plasma wasobtained and extracted as a above, with the methyl formate fractionsfrom the solid phase extraction being dried down for LC/MS/MS analysis.Values for ATLa₂ quantified in plasma are expressed in units of ng/mlplasma taken from the mouse, with n=3 for each time point.

RESULTS

Biostability of LX stable analogs. Following 3 h incubations of LXA₄ inmouse whole blood ex vivo, the predominant metabolite peak observed inthe LC/MS chromatogram of the extracted sample had a retention time andMS/MS spectrum matching that of 15-oxo-LXA₄, as generated by recombinant15-PGDH from synthetic LXA₄ (FIG. 1A). To determine whether addition ofbulky substituents to the native LX structure enhances biostability, twoaspirin-triggered lipoxin stable analogs, 15(R/S)-methyl-LXA₄ (ATLa₁)and 15-epi-16-(para-fluoro)-phenoxy-LXA₄ (ATLa₂), were incubated inmouse whole blood and compared to LXA₄. A methyl group at carbon-15 wasplaced as a racemate to protect both LXA₄ and 15-epi-LXA₄ in ATLa₁ and afluoride was placed at the para-position of the phenoxy ring of15-epi-16-phenoxy-LXA₄ in ATLa₂ (FIG. 1B). LC/MS/MS analysis of wholeblood incubations showed that ˜40% of LXA₄ was lost while both ATLa₁ andATLa₂ exhibited greater stability with ˜90% and ˜100% remaining,respectively (FIG. 1B). In human whole blood, quantitatively similarresults were obtained with ATLa₁.

Intravenous and local delivery of ATLa₂ inhibits TNF-a-induced PMNinfiltration in the dorsal air pouch. The six day murine dorsal airpouch is characterized by the presence of a nascent lining that enclosesthe air cavity and is composed of both fibroblast-like cells, which areindistinguishable from type B cells of murine knee synovium, andmacrophage-like cells, which share morphology with synovial type A cells(Edwards, J. C. W., Sedgwick, A. D. & Willoughby, D. A. (1981) J.Pathol. 134, 147-156). The air pouch therefore serves as an in vivomodel of the rheumatoid synovium and was used here to evaluate theimpact of intravenous and local delivery of ATLa₂ in the inhibition ofcytokine-mediated inflammation, and for direct comparison to the actionsof ASA and dexamethasone (Sin, Y. M., Sedgwick, A. D., Chea, E. P. &Willoughby, D. A. (1986) Ann. Rheum. Dis. 45, 873-877; Edwards, J. C.W., Sedgwick, A. D. & Willoughby, D. A. (1981) J. Pathol. 134, 147-156).Tumor necrosis factor-a (TNF-a) induces leukocyte infiltration,predominantly neutrophils (>75%), into the pouch with maximal cellaccumulation occurring between 2-4 h post-injection (Tessier, P. A.,Naccache, P. H., Clark-Lewis, I., Gladue, R. P., Neote, K. S. & McColl,S. R. (1997) J. Immunol. 159, 3595-3602). ATLa₂, dexamethasone, and ASAwere each injected locally into the air pouch of individual mice andimmediately prior to the administration of murine TNF-a. For systemicdelivery of ATLa₂, injections were given via the mouse tail vein beforelocal air pouch injection of murine TNF-a. Here, local delivery of TNF-aalone (20 ng/mouse) induced the recruitment of 4.8±1.1×10⁶ PMN into theair pouch at 4 h (FIG. 2). When ATLa₂ was delivered locally into the airpouch (10 mg/mouse), only 1.1±0.3×10⁶ PMN were present in the pouchexudate, representing ˜77% inhibition of the TNF-a-induced PMNinfiltration. Delivery of ATLa₂ (10 mg/mouse) by i.v. injection provedto be an even more potent method of inhibiting TNF-a-driven PMNinfiltration. The PMN recruitment values dropped to an average of7.9±2.9×10⁵ PMN/air pouch, representing an inhibition of ˜85%. Moreover,no apparent toxicity of ATLa₂ to the mice was observed. Localadministration of either ASA or dexamethasone also inhibited PMNrecruitment, but to a lesser extent than ATLa₂ by either local or i.v.delivery. An equivalent dose of dexamethasone (10 mg/mouse) led to 61%inhibition of PMN recruitment (infiltration of 1.7±0.5×10⁶ PMN), whereasa 100-fold greater dose of ASA (1.0 mg/mouse) was required to inhibitPMN infiltration to a similar degree as ATLa₂. The presence of1.5±0.6×10⁶ cells with 1.0 mg ASA represents 69% inhibition compared toTNF-a administration alone given to mice in parallel.

Histological analysis of the tissue lining surrounding the air pouchcavity showed that the addition of TNF-a resulted in a markedlyincreased number of neutrophils (FIG. 3A), which was reduced when ATLa₂was delivered by either intra-pouch injection (FIG. 3B) or i.v. via thetail vein (FIG. 3C) prior to TNF-a administration. Moreover, microscopicanalyses of dermal tissue from mice that received ATLa₂ treatment wereindistinguishable from those exposed only to vehicle (FIG. 3D), whichalso showed a mild neutrophil infiltrate accompanying this wound model.

ATLa₂ does not inhibit PMN recruitment by regulating vasoactivity. LXA₄exhibits both concentration- and vascular bed-dependent vasoactiveproperties. For example, topical administration of LXA₄ (1 mM) inducesarteriolar dilation in the hamster cheek with no change in venulardiameters while systemic delivery into rats produces a vasoconstrictorresponse in the mesenteric bed (26). In addition, 20 min infusion of 1or 2 mg/kg LXA₄ induces renal vasorelaxation in rats without changingmean arterial pressure (27). To determine whether the increasedstability of ATLa₂ enhances potential vasoreactivity at the therapeuticdose found to inhibit PMN infiltration in FIGS. 2 and 5, vascularchanges in response to ATLa₂ were compared directly to those ofIloprost, a prostacyclin stable analog that rapidly stimulates arterialvasodilation (Grant, S. M. & Goa, K. L. (1992) Drugs 43, 899-924). Addedto organ baths, ATLa₂ relaxed precontracted isolated rat aorta to ˜40%of the level of relaxation caused by equimolar treatment (1 mM) withIloprost (not shown). However, when 10 mg, or ˜24 nmol/mouse, of ATLa₂were injected into the tail vein as in FIG. 2, no apparent changes inmean arterial pressure were observed (FIG. 4). In sharp contrast,injection of equimolar quantities of Iloprost elicited a maximum meandecrease of ˜28 mmHg˜50 s post-injection, with pressure returning tobaseline after ˜8 min.

ATLa₂ inhibits PMN infiltration in murine ear skin to both exogenous andendogenous chemoattractants. Topical application of a racemic analogwith properties of both 15-epi-LXA₄ and native LXA₄, and 16-phenoxy-LXA₄(an analog of LXA₄) to mouse ear epidermis inhibits LTB₄-induced PMNinflux as well as vascular permeability changes (Takano, T., Clish, C.B., Gronert, K., Petasis, N. & Serhan, C. N. (1998) J. Clin. Invest.101, 819-826). Here, this ear skin model of inflammation was used todetermine whether i.v. or topical delivery of the whole blood stableATLa₂ could also inhibit PMN influx, which is maximal at 24 h aftertopical application of either LTB₄ or phorbol myristate acetate (PMA) toskin. Topical application of ATLa₂ inhibited both LTB₄-and PMA-inducedinflammation, by ˜78% and ˜49% respectively (FIG. 5). A single bolusi.v. injection of ATLa₂ (10 mg) did not inhibit PMN influx measured at24 h to either agonist applied topically to ear skin (FIG. 5) incontrast to i.v. and dorsal administration in the air pouch (FIG. 2).But, when i.v. injection of this analog was repeated at 20 h (4 h beforePMN measurement), LTB₄-induced PMN recruitment was inhibited by ˜22%(not shown).

ATLa₂ is rapidly cleared from plasma following i.v. injection. Sincei.v. tail vein delivery of ATLa₂ elicited a potent anti-inflammatoryresponse blocking PMN infiltration within a 4 h period in the dorsal airpouch (FIG. 2) but not at 24 h in the ear skin (FIG. 5), the questionarose as to what extent ATLa₂ possessed enhanced biostability incirculation following bolus tail vein injections. To address this, ATLa₂was extracted from mouse plasma collected at several time intervalsfollowing tail vein injections and the recovered materials werequantitated by LC/MS/MS. At 2 min post-injection,-34 ng/ml plasma weredetected. The levels of the analog decreased with time and were notdetected after 15 min. These results indicate rapid clearance from bloodand therefore rapid distribution and/or elimination (FIG. 6).

The fluorinated analog of 15-epi-LXA₄, ATLa₂, is a novel stable analoginhibitor of both direct (LTB₄) and indirect (TNF-a, PMA) actingchemoattractants. These in vivo observations further support the role ofthe aspirin-triggered lipoxin circuit as a novel and additionalmechanism underlying aspirin's anti-inflammatory therapeutic impact andprovide evidence for endogenous anti-inflammatory signaling pathways.

The present results indicate that specific design modifications of thenative LXA₄ structure, such as the addition of a C-15 methyl group(ATLa₁) or a bulky w-chain (para-fluoro)-phenoxy group (ATLa₂), prolongthe lifetime in blood of these compounds and therefore, potentially,their bioavailabilities as well. Such modifications sterically hinderconversion of the analogs, relative to rapid bioinactivation of thenative structure, by recombinant 15-PGDH in vitro (Serhan, C. N. (1997)Prostaglandins 53, 107-137). As evidenced by LC/MS/MS analyses, themajor product of this human dehydrogenase incubated with LXA₄ is15-oxo-LXA₄. LC/MS/MS analyses showed that 15-oxo-LXA₄ also was producedfrom LXA₄ in mouse whole blood (FIG. 1A), suggesting that the mouseshares with humans a common pathway for LXA₄ inactivation.

ATLa₂ proved to be a potent inhibitor of TNF-a-induced PMN infiltrationinto the air pouch cavity, as doses as low as 24 nmol/mouse deliveredlocally into the air pouch or by systemic i.v. injection via the tailvein resulted in ˜77% and ˜85% inhibition, respectively. Histologically,this wound model is thought to resemble rheumatoid synovium and TNF-ainjection initiates PMN recruitment to the cavity (FIG. 2) (Edwards, J.C. W., Sedgwick, A. D. & Willoughby, D. A. (1981) J. Pathol. 134,147-156). Injection of TNF-a into the air pouch increases, within thesurrounding tissue, C—C chemokine (murine monocyte chemotactic peptide-1and macrophage inflammatory protein-1a) and C—X—C chemokine (macrophageinflammatory protein-2) production and increases messenger RNA levelsfor the aforementioned chemokines as well as murine growth-relatedoncogene protein-a; all of which are collectively required forneutrophil recruitment (Tessier, P. A., Naccache, P. H., Clark-Lewis,I., Gladue, R. P., Neote, K. S. & McColl, S. R. (1997) J. Immunol. 159,3595-3602). Since ATLa₂ blocked TNF-a-induced PMN infiltration (FIG. 2),ATL disrupts this chemokine network in vivo. This finding may havetherapeutic implications, as a variety of pathological conditions,including rheumatoid arthritis, psoriasis, and Crohn's disease, haveassociated with them an over production of TNF-a, and therefore controlof this cytokine's actions is highly sought (Marriott, J. B., Westby, M.& Dalgleish, A. G. (1997) Drug Discovery Today 2, 273-282).

It was also found that ATLa₂ was more potent than ASA since a 100-foldgreater dose of ASA, delivered locally to the air pouch, resulted in alevel of inhibition of TNF-a-driven PMN recruitment that was less thanthat of ATLa₂. Furthermore, a locally administered, equivalent dose ofdexamethasone proved less potent as an inhibitor of PMN recruitment thanATLa₂ in this model. Given the unwanted side-effects associated with thestructures of both ASA (acidity that can lead to ulceration) anddexamethasone (steroid structure that can also impact physiologicsteroidal functions), structurally distinct compounds such as ATLanalogs designed on the basis of endogenous regulators of leukocytefunction may prove to be preferred therapeutic alternatives.

Applied topically to the ear, ATLa₂ also inhibited both LTB₄- andPMA-induced PMN recruitment, by ˜78% and ˜49%, respectively (FIG. 5).LXA₄ and ATLa₁ exhibit similar IC₅₀'s in vitro in the inhibition of PMNtransmigration across polarized epithelial monolayers or PMN adherenceto vascular endothelial cells (Takano, T., Clish, C. B., Gronert, K.,Petasis, N. & Serhan, C. N. (1998) J. Clin. Invest. 101, 819-826).Topical delivery of ATLa₁ in vivo inhibits LTB₄-induced PMN recruitment,but interestingly the level of inhibition afforded by the native LXA₄when added topically was less than 25% compared to that of either ATLa₁or ATLa₂ (Takano, T., Clish, C. B., Gronert, K., Petasis, N. & Serhan,C. N. (1998) J. Clin. Invest. 101, 819-826). These observationsregarding in vitro versus in vivo potencies between the analogs and thenative structure indicate that the ATL analogs posses enhancedbioavailability in vivo. Thus, in addition to protection from enzymaticinactivation, the structural modifications to the native LXA₄ structureincorporated in ATLa₁ and ATLa₂ also improved their topical delivery andcontributed to rapid distribution to tissue (FIG. 2).

Results obtained from the air pouch model, 4 h after administration ofthe analog, indicate that i.v. delivery of ATLa₂ to a remote site ofinflammation was surprisingly even more effective than topicalapplication. In sharp contrast are the findings with ear skin, wheretopical application of ATLa₂ elicited substantial inhibition oftopically applied pro-inflammatory mediators; i.v. delivery of theanalog showed no apparent inhibition of LTB₄-induced PMN recruitment. Itwas also found that the ATL analog was both stable ex vivo in wholeblood suspensions, with essentially completely quantitative recovery at3 h, and was rapidly cleared from plasma following i.v. injection intothe tail vein (between 15-50 min). Taken together, these results suggestthat ATLa₂ is rapidly distributed to tissues from i.v. injections,rather than eliminated, and could remain in an active form for severalhours, e.g. during the time course of the TNF-a-driven PMN recruitmentto the wounded dorsal pouch (FIG. 2). Furthermore, the absence of PMNinhibition through systemic delivery in the mouse ear model indicatesthat ATLa₂ displays site selective bioaction from circulation, such asto the dorsal pouch rather than to ear skin.

In summary, these results indicate that the inhibitory actions ofaspirin-triggered lipoxins are both tissue- and delivery site-dependentand are the first to show that stable analogs of ATL inhibit acuteinflammation at sites distant from the point of delivery. Since ATLstable analogs were designed as mimetics to incorporate the nativeaspirin-triggered structural features, the present findings, takentogether, provide new tools to examine endogenous anti-inflammatorypathways as well as avenues to approach the development of both topicaland intravenous anti-PMN therapies.

REFERENCES

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One of ordinary skill in the art will appreciate further features andadvantages of the invention based on the above-described embodiments.Accordingly, the invention is not to be limited by what has beenparticularly shown and described, except as indicated by the appendedclaims. All publications and references cited herein, including those inthe background section, are expressly incorporated herein by referencein their entirety.

1. A compound having the formula:

wherein X is R₁, OR₁, or SR₁; wherein R₁ is (i) a hydrogen atom; (ii) analkyl of 1 to 8 carbons atoms, inclusive, which may be straight chain orbranched; (iii) a cycloalkyl of 3 to 10 carbon atoms; (iv) an aralkyl of7 to 12 carbon atoms; (v) phenyl; (vi) substituted phenyl

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichmay be a straight chain or branched, and hydroxyl; (vii) a detectablelabel molecule; or (viii) a straight or branched chain alkenyl of 2 to 8carbon atoms, inclusive; wherein Q₁ is (C═O), SO₂ or (CN), provided whenQ₁ is CN, then X is absent; wherein R₄ is (a) H; (b) an alkyl of 1 to 6carbon atoms, inclusive, which may be a straight chain or branched;wherein R₅ is

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichmay be a straight chain or branched, and hydroxyl or a substituted orunsubstituted, branched or unbranched alkyl group; wherein R₆ is (a) H;(b) an alkyl from 1 to 4 carbon atoms, inclusive, straight chain orbranched; and pharmaceutically acceptable salts thereof excluding16-phenoxy-LXA₄ and 15-epi-16-(para-fluoro)-phenoxy-LXA₄.